GATA2 is required for lymphatic vessel valve development and maintenance.

Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functions of GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2 missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema

A non-canonical role for desmoglein-2 in endothelial cells: implications for neoangiogenesis

Desmogleins (DSG) are a family of cadherin adhesion proteins that were first identified in desmosomes and provide cardiomyocytes and epithelial cells with the junctional stability to tolerate mechanical stress. However, one member of this family, DSG2, is emerging as a protein with additional biological functions on a broader range of cells. Here we reveal that DSG2 is expressed by nondesmosome- forming human endothelial progenitor cells as well as their mature counterparts [endothelial cells (ECs)] in human tissue from healthy individuals and cancer patients. Analysis of normal blood and bone marrow showed that DSG2 is also expressed by CD34?CD45dim hematopoietic progenitor cells. An inability to detect other desmosomal components, i.e., DSG1, DSG3 and desmocollin (DSC)2/3, on these cells supports a solitary role for DSG2 outside of desmosomes. Functionally, we show that CD34?CD45dimDSG2? progenitor cells are multi-potent and pro-angiogenic in vitro. Using a ‘knockout-first’ approach, we generated a Dsg2 loss-of-function strain of mice (Dsg2lo/lo) and observed that, in response to reduced levels of Dsg2: (i) CD31? ECs in the pancreas are hypertrophic and exhibit altered morphology, (ii) bone marrowderived endothelial colony formation is impaired, (iii) ex vivo vascular sprouting from aortic rings is reduced, and (iv) vessel formation in vitro and in vivo is attenuated. Finally, knockdown of DSG2 in a human bone marrow EC line reveals a reduction in an in vitro angiogenesis assay as well as relocalisation of actin and VE-cadherin away from the cell junctions, reduced cell–cell adhesion and increased invasive properties by these cells. In summary, we have identified DSG2 expression in distinct progenitor cell subpopulations and show that, independent from its classical function as a component of desmosomes, this cadherin also plays a critical role in the vasculature.Lisa M. Ebert, Lih Y. Tan, M. Zahied Johan, Kay Khine Myo Min, Michaelia P. Cockshell, Kate A. Parham, Kelly L. Betterman, Paceman Szeto, Samantha Boyle, Lokugan Silva, Angela Peng, YouFang Zhang, Andrew Ruszkiewicz, Andrew C. W. Zannettino, Stan Gronthos, Simon Koblar, Natasha L. Harvey, Angel F. Lopez, Mark Shackleton, Claudine S. Bonde

In vitro assays using primary embryonic mouse lymphatic endothelial cells uncover key roles for FGFR1 signalling in lymphangiogenesis.

Despite the importance of blood vessels and lymphatic vessels during development and disease, the signalling pathways underpinning vessel construction remain poorly characterised. Primary mouse endothelial cells have traditionally proven difficult to culture and as a consequence, few assays have been developed to dissect gene function and signal transduction pathways in these cells ex vivo. Having established methodology for the purification, short-term culture and transfection of primary blood (BEC) and lymphatic (LEC) vascular endothelial cells isolated from embryonic mouse skin, we sought to optimise robust assays able to measure embryonic LEC proliferation, migration and three-dimensional tube forming ability in vitro. In the course of developing these assays using the pro-lymphangiogenic growth factors FGF2 and VEGF-C, we identified previously unrecognised roles for FGFR1 signalling in lymphangiogenesis. The small molecule FGF receptor tyrosine kinase inhibitor SU5402, but not inhibitors of VEGFR-2 (SU5416) or VEGFR-3 (MAZ51), inhibited FGF2 mediated LEC proliferation, demonstrating that FGF2 promotes proliferation directly via FGF receptors and independently of VEGF receptors in primary embryonic LEC. Further investigation revealed that FGFR1 was by far the predominant FGF receptor expressed by primary embryonic LEC and correspondingly, siRNA-mediated FGFR1 knockdown abrogated FGF2 mediated LEC proliferation. While FGF2 potently promoted LEC proliferation and migration, three dimensional tube formation assays revealed that VEGF-C primarily promoted LEC sprouting and elongation, illustrating that FGF2 and VEGF-C play distinct, cooperative roles in lymphatic vascular morphogenesis. These assays therefore provide useful tools able to dissect gene function in cellular events important for lymphangiogenesis and implicate FGFR1 as a key player in developmental lymphangiogenesis in vivo

FGF2 and VEGF-C promote tube formation of primary mouse LEC.

<p>(a) Primary LEC were cultured for 24 h and imaged immediately following the addition of Matrigel. (b) Primary LEC were cultured for 24 h followed by addition of Matrigel alone or Matrigel containing FGF2 (10 ng ml⁻¹), VEGF-C (200 ng ml⁻¹) or a combination of FGF2 and VEGF-C. Images were captured after a further 48 hours. (c) Primary LEC were cultured for 24 h followed by addition of Matrigel containing FGF2 (10 ng ml⁻¹) or a combination of FGF2 (10 ng ml⁻¹) and VEGF-C (200 ng ml⁻¹) and tyrosine kinase inhibitors SU5402 (10 µM, FGFR1), SU5416 (5 µM, VEGFR-2) or MAZ51 (5 µM, VEGFR-3). Three replicates of each treatment were performed and images are representative of at least three independent cell isolations. Inset panels in (c) illustrate magnified views of boxed regions. Scale bars represent 250 µm. Quantification of average vessel diameter (d) using Lymphatic Vessel Analysis Protocol (LVAP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040497#pone.0040497-Shayan1" target="_blank">[28]</a> and ImageJ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040497#pone.0040497-Abramoff1" target="_blank">[29]</a> software and branch points per well (e) using AngioTool software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040497#pone.0040497-Zudaire1" target="_blank">[30]</a>, for each treatment indicated. Data show mean ± s.e.m. and are derived from 2 independent cell isolations, each prepared from multiple litters of embryos, and 3 replicates of each treatment (n = 6). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

FGF2 stimulates primary mouse LEC proliferation.

<p>(a) Primary LEC were cultured in EBM-2+0.5 mg ml⁻¹ Albumax (Control) or EBM-2+0.5 mg ml⁻¹ Albumax containing FGF2 or VEGFC at the indicated concentrations for 48 h. LEC proliferation was measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). Data shown represent mean ± s.e.m. and are derived from 3 independent cell isolations, each prepared from multiple litters of embryos, and 5 replicates of each treatment (n = 15). (b) FGF2 stimulated LEC proliferation is inhibited by an FGFR tyrosine kinase inhibitor but not by VEGFR inhibitors. Primary LEC were cultured in EBM-2+0.5 mg ml⁻¹ Albumax (Control), or EBM-2+0.5 mg ml⁻¹ Albumax and FGF2 (10 ng ml⁻¹), together with the tyrosine kinase inhibitors SU5402 (10 µM, FGFR inhibitor), SU5416 (5 µM, VEGFR-2 inhibitor) or MAZ51 (5 µM, VEGFR-3 inhibitor). LEC proliferation was measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). Data shown represent mean ± s.e.m. and are derived from 3 independent cell isolations prepared from multiple litters of embryos and 5 replicates of each treatment (n = 15). **<i>P</i><0.01 ***<i>P</i><0.001.</p

FGF2 and VEGF-C promote migration of primary mouse LEC.

<p>(a) Confluent monolayers of primary LEC were scratched and cultured in EBM-2+0.5% FBS (Control), or EBM-2+0.5% FBS containing FGF2 (10 ng ml⁻¹) ± SU5402 (10 µM) or VEGF-C (200 ng ml⁻¹) for 8 h. Dotted white lines mark the boundaries of the wound at 0 h. Scale bars represent 125 µm. (b) Quantification of area migrated in 8 h. Data represent mean ± s.e.m. and are derived from 3 independent cell isolations, each prepared from multiple litters of embryos, and 5 replicates of each treatment (n = 15). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p